Abstract

Substantial data implicate the commensal flora as triggers for the initiation of enteric inflammation or inflammatory disease relapse. We have shown that enteric epithelia under metabolic stress respond to nonpathogenic bacteria by increases in epithelial paracellular permeability and bacterial translocation. Here we assessed the structural basis of these findings. Confluent filter-grown monolayers of the human colonic T84 epithelial cell line were treated with 0.1 mM dinitrophenol (which uncouples oxidative phosphorylation) and noninvasive, nonpathogenic Escherichia coli (strain HB101, 10(6) CFU) with or without pretreatment with various pharmacological agents. At 24 h later, apoptosis, tight-junction protein expression, transepithelial resistance (TER; a marker of paracellular permeability), and bacterial internalization and translocation were assessed. Treatment with stabilizers of microtubules (i.e., colchicine), microfilaments (i.e., jasplakinolide) and clathrin-coated pit endocytosis (i.e., phenylarsine oxide) all failed to block DNP+E. coli HB101-induced reductions in TER but effectively prevented bacterial internalization and translocation. Neither the TER defect nor the enhanced bacterial translocations were a consequence of increased apoptosis. These data show that epithelial paracellular and transcellular (i.e., bacterial internalization) permeation pathways are controlled by different mechanisms. Thus, epithelia under metabolic stress increase their endocytotic activity that can result in a microtubule-, microfilament-dependent internalization and transcytosis of bacteria. We speculate that similar events in vivo would allow excess unprocessed antigen and bacteria into the mucosa and could evoke an inflammatory response by, for example, the activation of resident or recruited immune cells.

Low-dose DNP does not affect E. coli HB101 growth but does reduce epithelial ATP content. (A) Comparison of E. coli HB101 to enterohemorrhagic E. coli, where only the latter was found to have abundant pili (arrowhead) and sex pili (arrows). (B) Representative bacterial growth curve (one experiment of three) showing that 0.1 mM DNP does not affect E. coli growth. (C) The same dose of DNP results in a significant decrease in T84 cell ATP levels by 4 h posttreatment (n= three to five experiments/time point with two to three epithelial preparations/experiment; ATP was normalized to time-matched naive controls; ✽, P < 0.05 compared to control as determined by ANOVA, followed by pairwise Student t test comparisons).

Exposure to E. coli HB101 with or without DNP results in altered expression of TJ proteins. (A) Representative immunoblots show the reduced expression of TJ proteins 24 h after culture with DNP (0.1 mM) and E. coli HB101 (106 CFU/ml). Expression of claudin-1, -2, and -4 was not consistently altered (n = 2 to 5). Equal amounts of protein were loaded, and the blots were reprobed for claudin-4 (whose expression was unaltered by any treatment), and total protein staining of the gels (not shown). (B) Densitometric analysis (arbitrary units) reveals that although exposure to DNP or E. coli alone can reduce the expression of specific TJ proteins, such reductions were only statistically significant for actin (n = 4), occludin (n = 6), and ZO-1 (n = 6) when the epithelial were exposed to DNP and E. coli (✽, P < 0.05 compared to control naive T84 cell preparations as determined by ANOVA).

Interference with microfilament assembly reduces both E. coli HB101 internalization and translocation in DNP-cotreated epithelia. (A) Jasplakinolide treatment of T84 cell monolayers exposed to DNP (0.1 mM) and E. coli (106 CFU) significantly reduces bacterial internalization (mean ± the SEM; n = 4 to 12 monolayers from three to four separate experiments; ✽, P < 0.05 compared to DNP and E. coli only [i.e., zero jasplakinolide]). (B) The reduced internalization of the E. coli is accompanied by reduced translocation into the basal compartment of the culture well (numbers inside the bars indicate the number of T84 cell monolayers positive for bacterial translocation compared to the total number of epithelial preparations observed). The inset shows a representative plate showing the lack of bacterial colony growth from medium obtained from the basolateral culture well of epithelia treated with jasplakinolide (J) or cytochalasin D (Cyto D; 0.2 μΜ). Each spot is from a separate epithelial monolayer.